1. Technical Field
The present disclosure relates to an analyzer for biochemical analyses and to a method for determining concentrations of fluorescent substances in a solution.
2. Description of the Related Art
As is known, the analysis of nucleic acids includes, according to different modalities, preliminary steps of preparation of a specimen of biological material, amplification of the nucleic material contained therein, and hybridization of individual target or reference strands, corresponding to the sequences sought. Hybridization takes place (and the test yields a positive outcome) if the specimen contains strands complementary to the target strands.
At the end of the preparatory steps, the specimen is examined for checking whether hybridization has taken place (the so-called “detection step”).
Several inspection methods and apparatuses are known for this purpose, for example of an optical or electrical type. In particular, the methods and apparatuses of an optical type are frequently based upon the phenomenon of fluorescence. The reactions of amplification and hybridization are carried out in such a way that the hybridized strands, contained in a detection chamber made in a substrate, include fluorescent molecules or fluorophores (the hybridized strands may be fixed to the bottom of the detection chamber or else remain in liquid suspension). The substrate is exposed to a light source having an appropriate spectrum of emission such as to excite the fluorophores. In turn, the excited fluorophores emit a secondary radiation at an emission wavelength greater than the peak of the excitation spectrum. The light emitted by the fluorophores is collected and detected by an optical sensor. In order to eliminate the background luminous radiation, representing a source of disturbance, the optical sensor is provided with band-pass filters centered at the emission wavelength of the fluorophores.
The detection of different substances in one and the same specimen requires as a rule the use of distinct fluorophores, having respective excitation and emission wavelengths. Various sets of optical filters must hence be coupled in succession to the light source and to the optical sensor for analyzing the responses in the excitation and emission bands of each fluorophore.
A limitation of known systems depends upon the need to envisage a mechanism of replacement of the filters, without which the analyses could not be conducted automatically. Mechanisms of this sort may comprise one or more carousels, mounted on which are the filters, and respective motors controlled to couple the pair of filters to the light source and to the optical sensor. This need, however, entails considerable overall dimensions, preventing production of independent portable analyzers.
Alternatively, it is possible to use multiple-band filters, but solutions of this type usually penalize the precision of detection. The excitation and emission bands of fluorophores of different types are in fact centered around different wavelengths, but have significant and partially overlapping tails. The optical multiple-band filters are in general less selective and are far from effective in preventing phenomena of mutual interference (referred to also as “crosstalk”). On account of the poor selectivity of multiple-band filters, in practice, the fluorophores can be excited also by stimuli of the excitation band of a different fluorophore and the optical sensor may collect light emitted by fluorophores different from those which are specifically excited (i.e., excited by tails of bands different from their own).